Method of producing with bacteria poisons intended for immunizing purposes



?ateniesl liiiar. 3923. Y

narrator) en runes .1 "L135, IEJIABIY, ASSIGNGR, BZ MESNE ASSIGNEENTS,

LQUNDA'XEGEM, Ili'fii, A CORPDEATZON OF DELAWAEE.

WING WITH BACTERIA POISQNS INTENDED FOR IMMUNIZING PURPQSEE.

No Brawing.

To all whom, it may; 1-,

Be it known that i r lininnnnnone, residing: at 1'? S bel c, Berlin-Charlottenburg, Germany. has invented certain new 5 and useful lmprm cmcnts in h'lethods of Producing with Harte for ln'ununizino Purposes, of which the following is a specification.

My invention relates to immunizing me- 19 die and has for an object a inethcd for the production of a bacterial poison or virus suitable for immunizing purposes.

The invention in brief comprises treating a dense suspension of bacteria with ambeceptor and complement, and separating after some time the toxic liquid from the precipitate'by any suitable means such as, for ex ample, centrifuging. in accordance with the method a toxin or virus is obtained which is eflicacious, for the setting up of active and passive immuity in an individual. The toxin obtained is the same as that which may be formed within the individual during the natural metabolist-ic processes and it is therefore particularly suitable for devitalizing bacteria and for nullifying their action.

Another object of the invention is to produce a poison which may be administered in exact predetermined-anddesired quantity and quality with respect to the nature of the disease.

A further object of the invention is to render the product obtained stable at practicallv any temperature and free from germs, 3

Up to the present the method generally used for producingan immunizing agent or a vaccine cohsists in inoculating an individual with bacteria. the assimilation or disassimilation products of bacteria (mains), dissolved substances derived from the body of ani als, etc. Further. it has been pro posed' of! Folia Serologica, Vol. L, page 428, sub No. 428) to digest bacteria with gastric juice and to use the partially diested bacterial mass, after in vacuo, as a vaccine. Such methods are based on the principle that the bacteria contain primar toxic substances which when set free wit in the organism efiect the P01- somng. I I

In" contradiction to the above I hare discovered as a result of many experiments 1 1a Poisons Intended son, i.

Zeitschrift fur Imrnunitatsforschuni; und

concentration truncation filc February 6, 1915. Serial Lto. 6, 535.

that by the interaction of the amboceptors and complementary substances contained in the body of a being and the bacteria which may have come into said bod; a new poison 1S originated. This is rendered very. prohable by the fact that quiteanalogously act: mg poisons of very great eiiiciency are separated from. amorphous albumen, for example, egg-albumem or from animal cells for instance, so-called dark spots or shadows in blood globules, Whichof itself is not poisonous at all. Clo obtain such a virus the albumen is left for a period of time in a suitable receptacle in contact with an homoh ogous antagonist, centrifuged and the residue thus obtained is subjected to the action of a serum containing a complement. The liquid is separated from the residue and contains the above mentioned virus or poie.., the so-called anuphylatoiiine (cf.

Experimentclle Therapiei 1st part, original Volume IV, page 636 and fclloyving l The quantity of bacteria suiiicient for the preparation of a. dose oi the alcove men-' tioned poison capable of exerting an acute killing action, if contained in a test-tube under the proper conditions, is under certain circumstances many times smaller than the dose. of the same bacteria which on being directly injected in the body gives rise to the same irillin effect. This may be due to the fact that the formation'ot toxin in the body of: the animal into which bacteria have heen injected is so slow that at any given moment dosage of the poison suf ficient for exerting a killing" action is not present, and that the poison, which for the time being has been produced may be modified and converted into non-poisonous modifloat-ions.

In accordance with my method, bacteria in a dense suspension are treated with. a small quantity of amboceptor and its conr clement, and the li uid containing the immunizing ncison, which is highly toxic, separated from the precipitate, forexample, by means of a centrifuge.

In carrying out my invention the following method may be used:

To a small quantih (colony) of typhus bacteria (i. e. 2 milligrams) suspended in a: cubic centimeters of a physloiiogical solution of common salt is added a small quantity (0.1 com.) of a serum conliaining amhocepfsor and after stranding iornhous 24: hours iii; the temperature of an icewhesb the mass is centrifuged. To the sediment, containing bacteria and emboeeptor, which is freed by repeated lixiviubion with r. physiological solution of common sell: from iihe lust traces of serum, are added 4 com. of fresh normal cobuys-serum containing a complement. After siandingfor several hours at the temeraturc of on ice-chest during which'time it, is frequenbl shulzemihe mess is again centrifuged. serum into the blood-current of a colon-ya weighing about 200 g., is is ascertained that by the contact with the bsceerischsirged with nmbocepboi-i the cobayusei'umhas obtained such a toxicity that it is capable of killing on animal of the ssniegenus in a few minutes and convulsions are induced. In orderio prove that the. proportion or'percentage of complement in the poison or virus is importent, control-tests have shown that when cobuye-serum is heated to 58 so as to be ireed from. its content. of complement the does not. reduce poison with bacteria containing em oceptor. However the conmoi at the temperature dunes the poison more quickly than at nor i of the v mel body icemperature.

In the method forming the objecr presenl, invention small quantities of beeteriu are unsuitable, even when the latter ere used in the reaction process in such a conceniiretion that they are not completely killed by the amboccptor. it is necessary so operate with e high concentration of suspended bacteria in order to obtain a suitable immunizing virus or poison. Further, only small quantities of ambecepizor are required, as no killing of the bacteria is intended, so that the proportion of ambocepior contained in normal serum is quite suliicient and the contact of the bacierie with the nori'nal serum suffices, because normal serum contains besides embocepilor the necessary complement. i

The method herein. described aim be prepare toxic substances and note bueticriolysis which was formerly the objectof the known processes and would have a deleterious of feet. Also dead bacteria may be used without diiiiculisy and Wiifill exactly the samercsult. 9n account of the fact that the method of producing the new poison corresponds to the reactions taking place in the organism. is can. be used with greater advantage for uei'ive and passive immunizing in certain diseases, as for example byphus, izuloercu losis, cholera, epidemic diseases ofunimals and the like. The immunizingsubstances when they are injected or inoculated into on individual produce an increasing or silreng'ihening of the action which fishes in she organism itself. Thus ssid subpon injecting ihe cobeyu of an ice-chest, promesses stances perform a part of the work which is to be performed by the similar natural immunizing substances which are normally formed in the body. The natural immunizing action, which takes place is highly assisted by'ihe novel immunizing substance. Said immunizing substance acts therefore in a quite, rational Way in contradiction to those previously known. The ,former rely upon posterior or subsequent acfsiens inthe body, which are beyond the ontrol of the administrator. They are never free from subsequent undesired or noxiousactions, which never occur with my new substance, inasmuch as'it can not only "be injected in exactly. predetermined quantities, but also in a given state of reaction, according to the special renditions observed during, production. Consequently, said poisons can also be used for producing antitoxin serum by treating animals with the poisons, after which the serum is obtained in the usual way by blood withdrawal and clotting I am aware that Buchner & Di'eudonn (cf. Zentralblaii: fur Bakteriolo'gie VI 1889,

page 10,2111. 2 and Dieudpnn, Immunitut, Schutzimpfung u. Serumtherapie, 1909 page 35-38 und 211) have reported methods speoially adopted for diagnostic purposes, in which highly diluted suspensions of bacteria (a quantity of 2 milligram of an 18 'hour sgsrculture in bouillon in the proportion of 1250800 or of e 2 ihour bouillon culture 5000 times diluted with bouillon) in fresh blood or serum are brought into contscl; with a great quantity of emboceptor and a certain quantity of complement. By these experiments only the bactericidal property of-rhe serum for special Said method operated with a dilution of 1:500000, whereas in the present method according to the example given typhus bacteria in a quantity of 2 mg. (taken up with the eyelet formed at the end of a wire) are used and treated with 4 ccm. serum. Therefore the concentration is very much higher. Practical experiments have shown 'thnt thencw toxic liquids which arenot'very stable may be converted by drying at low temperatures, preferably at temperatures below 40". into dry substances which in a. desiccator and in the dark may be conserved during a long time. In addition I have found that the dry substances can be licud'd to about 100 without becoming inactive. whilst the liquid becomes .inactive at :1 temperature of about The (11 substances may be dissolved in correspon ing quantities of water .Without: residue. This behaviour of the immunizing media obtainable according to the process forming rho object of the present invenilion is of veins, because it is possible in this manner cases was ascerrained, as the concentrations were very low I and the amount of emboceptor very high.

1 I claim: I

not only to bring the substances in question 8. A method of producing immunizing into a suitable form for transport and conmedia which comprises treating dead. bacservation, but also to render them free from teria with amboceptor and complementand germs. A sterilizing may take place during then separating the resultant precipitate the production by heating the bacteria from the toxic li 'uid. charged with amboceptorto about 100,that 9. A method 0 producing immunizing is, the bacteria. which have been subjected media which comprises treating a dense susto the first step of the method. The sterilipension of dead bacteria with normal sezation does not effect the further treatment rum, separating the resultant precipitate with complement and the quality of the from the toxic'liquid, drying said liquid. at

a low temperature and sterilizing the dry residue at a higher temperature. 1. A method of producing. immunizing A method of producing immunizing media which comprises treating a dense susmedia'which comprises suspending a colony poison or virus is not impaired.

"pension of bacteria with amboceptor and of bacteria in a physiological solution of with 'a' serum containinga complement, and common salt, adding thereto a serum conprecipitate from taining an amboceptor, maintaining the mass for several hours at the temperature 0: an

ice-chest, centrifuging the mass, dding to separating. the resultant the toxic iquid.

- 2. A method of producing immunizing media, which comprises treating a dense the residue containing the amboceptor asesuspension of bacteria with 'a'mboceptor and rum containing a complement, maintainlng with a serum containing a complement, se'pathe mass formed at the temperature of an 'ratin the resultant precipitate by centrifugice-chest for several hours and shaking the ing mm the toxic liquid, and drying. said liquid at a low temperature.

3. "A method of producing immimizing rate the toxic liquid from the sediment. media whichcomprises treating a dense sus- 11. A method for producing immunizing pension "of bacteria with amboceptor and media which comprises suspending a colony complement,'separating the precipitate from of bacteria in a physiological solution of the toxic liquid fhycentnfriging, drying chmmon salt, adding thereto a serum consa'id liquid at a low temperaturgand steriliztaining an ambooeptor, maintaining the mass ing the dry residue ate. higher temperature. for several hours at the temperature of an 4. A'method. of producing immunizing ice'-chest centrifuging the mass, liniviating media which comprises treating a dense susthe resulting residue containing an embopension of bacteria with amboceptor, sterilceptor, adding to the sediment a serum conizing the mass, then adding a serum containtaming'a complement, maintaining the mass ing a complement and separating the pre- "for several hours at the temperature of an cipitate by suitable means from the toxicsice chest, shaking the mass periodically durliquid. ing the maintenance, again centriiuging the 5. A method of producing immunizing s'eid mass to separate the toxic liquid from media which comprises treating a dense susthe sediment and then solidifying the liquid. pension'of bacteria with amboceptor, heat- 12. A- method of producing an antitoxic: ing the mass to a temperature of about 100 serum, which comprises treating a/dense sus- (1, then adding a. serum containing a compension of bacteria with amhoceptor and plement, and 'separating the precipitate from with a serum containing a complement, separating the precipitate by suitable means mass periodically during the exposure, and

the toxic liquid. a

6. A method of produc1ng immunizing from the toxic liquid containing the immumedia which comprises treating a densesusnizmg poison, then'lnoculating an animal pension of bacteria with amboceptor, heatwith saidimmunizin'g poison, and extracting ing the mass to a temperature of about 100 the obtained serum from the body of" the C. then adding a serum containing a compleanimal.

again centrifuging the said mass to 'sepa-- ment and separating the precipitate from the toxic liquid by centrifuging.

7. A. method of producing immunizing media which comprises treating a dense suspension of bacteria with normal serum and separating the resultant precipitate from the toxic liquid.

In testimony whereof I aiiix my signature in presence of two witnesses.

ERN"ST FRILDBERGER. Witnesses:

M. Wanna, G. Bower.

lit) 

